Development and Manufacture of Therapeutic Bispecific Antibodies

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Glycosylated bispecific immunoglobulin antibodies — heavy chains in green and pink and light chains in blue and yellow — engineered to target two different antigens. (HTTPS://STOCK.ADOBE.COM)

To meet the ongoing need for new and improved drugs, the biopharmaceutical community strives to create molecules with new functions. Bispecific antibodies (bsAbs), which can simultaneously home in on two different targets, illustrate the scientific ingenuity needed for this task. The basic proof of concept for these complex molecules was established in 1960 (1), and their application to the redirection of effector cells was reported in the mid-1980s (2–4), but producing them has proved to be challenging.

Many technical advances, both large and small, were needed before bsAbs could be prepared in quantities that would be sufficient for their clinical development. Key among these advances was the invention of the single-chain variable fragment (scFv) and the creation of methods that could enable antibody protein chains to form two different target binding sites rather than two identical ones, as occurs in naturally produced antibodies (5). A T-cell–redirecting bsAb composed of two different scFvs was produced in the mid-1990s (6). The “knobs-into-holes” method of engineering antibody domains for heavy chain heterodimerization debuted about the same time (7). Bispecifics were constructed with two different heavy chains and two identical light chains in 1998 (8). Building on those early advances, antibody engineers since have designed more than 100 types of bsAbs (9–11).

To date, four bsAbs have been granted marketing approvals. Early scientific discoveries relating to scFvs and T-cell redirection served as a foundation for development of the first two approved products, Removab (catumaxomab) and Blincyto (blinatumomab). Catumaxomab was first approved in Europe for malignant ascites in 2009 but subsequently withdrawn for commercial reasons. By contrast, the 2014 approval of blinatumomab, which comprises a tandem scFv targeting CD19 and CD3 on T cells, sparked substantial interest in the T-cell redirection approach. The more recently approved bsAbs — Hemlibra (emicizumab) and Rybrevant (amivantamab) — rely on different designs. The former’s bispecificity derives from the common light-chain approach; the latter was constructed using Genmab’s proprietary DuoBody technology. By that method, two antibodies (each with differing specificity) are expressed individually, then purified and mixed together under conditions that favor formation of a bispecific molecule.

Clinical Pipeline
Since 2000, more than 200 bsAbs sponsored by commercial entities have been evaluated in clinical studies, although most of them have entered clinical study only relatively recently. That is not surprising given the industry’s relative inexperience with methods available early on. BsAbs entered clinical study at a rate of only about one per year during the 2000s, increasing to about six per year in the first half of the 2010s. Successes and expansion in construction and production methods, however, have enabled remarkable growth in the pipeline since then. In the past six years, an average of 27 bsAbs have entered clinical study each year. As a consequence of that growth, the commercial clinical pipeline now includes ~160 bsAb-based therapeutics. Most are in early stage development, with only about a dozen in late-stage clinical studies).

The commercial clinical pipeline of bsAbs is focused primarily on cancer treatments. Of the ~160 bispecifics in clinical studies, about 90% are undergoing evaluation for one or more types of cancer. Two approaches dominate: T-cell engagement and immune-checkpoint modulation. They respectively make up ~47% and ~28% of the anti-cancer bispecifics currently in clinical trials. T-cell engaging bispecifics such as blinatumomab target a tumor-associated antigen (TAA) and CD3 on T cells, thereby bringing cytotoxic T cells into proximity with tumor cells. Of the bispecifics that target CD3, the two most frequent TAA targets are B-cell maturation antigen (BCMA) and CD20. Of the bispecific immune-checkpoint modulators, common targets include PD-1 and PD-L1, CTLA-4, 4-1BB, OX40, TIM3, TIGIT, LAG-3, and CD47.

Manufacturing Requirements
Development of suitable biomanufacturing processes is a core element of commercializing new bsAb candidates. Companies defining processes for their bispecific candidates invariably want to maximize productivity using expression systems and upstream processes that generate high titers coupled with efficient purification processes that maximize yield while providing required purity. Such processes must be robust and meet the same regulatory requirements as other biological medicines do.
Developers of bsAbs have noted that production scales for these molecules often match those of standard monoclonal antibodies (MAbs) (13). We find that bsAb sponsors generally prefer manufacturing early phase clinical material at smaller scales than are common for conventional MAb production because of higher potencies that provide for lower dosing requirements. Nevertheless, companies could scale up their processes to large cell-culture production facilities for late-phase clinical studies and commercialization. Scalability of the production processes for next-generation antibodies is as important as it is for standard MAbs.

Platform manufacturing processes often are highly desirable for companies with pipelines that include multiple bispecific candidates. Such approaches enable rapid early stage process development; provide time and cost efficiencies; reduce raw-material and consumables lead-times; and facilitate process transfers, analytical development, and regulatory filings. A platform approach typically is adopted when candidates are structurally similar, but it also can be applied to disparate molecules. For example, Bristol-Myers Squibb (BMS) implemented a platform for bsAbs despite the candidates’ having different structures and formats (14).

Expression Systems
Single-chain variable fragments have been expressed in bacterial, yeast, mammalian-cell, insect-cell (baculovirus based), plant-cell, and cell-free expression systems. Escherichia coli is a common choice for expressing scFv proteins because bacteria grow quickly on inexpensive media and can produce proteins in large quantities. However, expressed scFv molecules can be misfolded or form inclusion bodies that require additional downstream processing steps to solubilize and refold proteins.

Using mammalian cell lines such as Chinese hamster ovary (CHO) cells for scFv production can overcome those problems because of the advanced internal protein-folding processes in eukaryotic cells and their ability to perform complex posttranslational modifications. Often used for biomanufacturing of MAbs and many other proteins, highly scalable CHO cell cultures can express proteins stably and at sufficiently high titers to compete with prokaryotic expression systems (12). Mammalian cell lines grow more slowly than prokaryotes do, however, making production take longer.

IgG-like bsAbs are expressed predominantly in mammalian cell lines for the same reasons. However, a manufacturing challenge arises in ensuring the correct assembly of antibody fragments into IgG-like bispecifics. Four different polypeptide chains can be combined in 16 possible ways, only two of which (~12.5% of what is expressed) have a desired asymmetric heterodimeric structure, with the rest (~87.5%) remaining as impurities (12).

That problem has been evident particularly with the quadroma technology used to produce some of the first bsAbs. Quadromas are created from the fusion of two hybridoma cell lines that express monospecific, bivalent antibodies. The resulting cells produce heavy and light chains from both parent lines. The molecules assemble randomly, so only a small proportion of them form the desired bsAbs. The others become product-related variants, including free heavy chains, light chains, homodimers, half molecules, and mispaired antibodies that are impurities to remove through a complex purification process. The combination of poor expression levels for desired antibody structures and intensive purification schemes ultimately result in low overall process productivities (10).

Protein/Cell Engineering Improves Manufacturability
To address that challenge, protein engineers developed a number of different strategies for designing bsAb molecules with improved manufacturability characteristics. Fc heterodimerization techniques ensure that asymmetric molecules are produced through a combination of complementary heavy chains, which substantially reduces the number of combinatorial possibilities (16). The knobs-into-holes technology enables Fc heterodimerization and reportedly improves antibody stability, making proteins with improved tolerance for the types of pH conditions that fluctuate during purification by protein A chromatography (17). CrossMab technology (15) and the common light-chain method are protein-engineering approaches that reduce further the number of structural combinations produced by ensuring correct pairings of light chain and heavy chains (16).

Such methods have enabled biopharmaceutical companies to use production schemes for bispecific candidates that are very similar to those used to make MAbs. For example, Merus has manufactured a bispecific, humanized IgG1 antibody (zenocutuzomab) at 2,000-L scale using a DG44 CHO cell line with a cell culture process that took 14 days in a production bioreactor. A familiar downstream process for MAbs was used to purify the bulk drug substance, with a protein A capture step followed by low-pH hold for virus inactivation, flow-through anion-exchange chromatography, and a final cation-exchange chromatography step to remove minor residual homodimers and half-IgG contaminants. Eluate was filtered through a 20-nm virus-removal filter before solution concentration and drug-substance formulation to 20 g/L. The Merus process provided bioreactor titers in the range of 1.0–1.5 g/L and downstream yields of 60–75% (16). Although such process performance is not especially high by MAb standards, it would be considered acceptable for many such products in early phase development.

Despite improvements in candidate design that lead to forced pairings, generating cell lines that produce heterodimers at high concentrations without simultaneous increases in product-related impurities remains an important goal in the development of economically viable processes that will yield safe and efficacious products (17).

One group of cell-line engineers have defined a process for generating cell lines that express bsAbs comprising three polypeptide chains (18). To increase the likelihood of correct protein assembly, the scientists introduced three genes into separate vectors, enabling developers to optimize their ratios of the different plasmids that resulted. Transfected pools were analyzed with a cell-secretion assay to determine which of them contained high proportions of high producers. The team used capillary electrophoresis with sodium-dodecyl sulfate (CE-SDS) to identify the pool containing the highest proportion of a desired bispecific heterodimer form. Clones were created from the pool with the most optimized plasmid ratio and grown in microscale bioreactors to assess growth performance, productivity, and heterodimer purity. Ultimately the team selected a clone that expressed heterodimers to 97% purity on CE-SDS and 98% purity by size-exclusion high-performance liquid chromatography (SEC-HPLC) with <2% aggregates. The resulting cell line expressed a bsAb at ~5 g/L and proved to be stable beyond 60 generations (18).

Not all development groups have adopted the approach of integrating each gene coding for a bsAb into separate vectors. A team from Eli Lilly integrated two heavy-chain and two light-chain genes into a single-plasmid quad vector that gave titers comparable to multiplasmid systems while facilitating generation of stable clones (19).

Production and Processing
Upstream: Because the composition of cell culture media has been shown to affect product quality, media design should be considered as a means to control levels of partial products, aggregates, and product variants in cell culture harvests (17). Scientists at Amgen have shown that cell culture temperature can modulate half-antibody and aggregate formation in a CHO cell line. The team reduced product-related impurity formation by adopting a biphasic culture process with a growth phase at 36 °C followed by a cooler phase that improved product quality to increase yields through the downstream process (20).

Downstream: BMS has used a standard three-stage filtration process in harvesting cell cultures with its bsAb platform. The process includes a protein A affinity capture step, but the team performed additional development work to optimize loading capacities and maximize step yields. Elution-buffer screening helped optimize yields and minimize aggregation. Product-related impurities were removed effectively following two further polishing steps of ion-exchange and mixed-mode chromatography. Although step yields across one or two polishing steps were lower than might be expected for a MAb, the platform allowed for purification of three different bsAbs to meet product-quality regulatory requirements for clinical trials (14).

Because of fundamental structural similarities between monoclonal and bispecific antibodies, many current purification methods for bispecifics are based on established purification processes for conventional MAbs. Affinity, charge, size, hydrophobicity, and mixed modes of chromatography often are used, with additional strategies applied to overcome the unique challenges presented by bsAbs (21). Those include aggregation, fragment formation, and mispaired products.

For example, researchers at Regeneron have used differential protein A chromatography to remove homodimeric heavy-chain mispaired molecules after introducing a point mutation into the Fc region of one heavy chain to prevent protein A binding. Homodimers with the mutation thus flow through the capture column during loading. Fully bsAbs can be separated from those without the mutation by leveraging their decreased avidity for the protein A matrix (22).

Although protein A chromatography still might be the primary means for bsAb capture, TeneoBio and its partners have reported replacing that affinity resin in the capture step for a bispecific CD3-TAA candidate with a another based on a llama VHH “nanobody” fragment immobilized on agarose. The ligand binds specifically to the CH1 domain of IgG heavy chains. That enabled the team to reduce copurification of product variants with intact Fc domains and also to elute the product under mild conditions that minimize the aggregation of both active and inactive variants (23).

Moving beyond the initial capture step, another group of purification-development scientists have adapted a mixed-mode polishing step to facilitate removal of a by-product resulting from incomplete chain pairing of a symmetric bsAb (24). This is accomplished by operating the column in a weak partitioning mode, which provides for high throughput, good yield, and effective by-product removal (24).

Pushing Antibody Technologies Forward
Generating antibodies with two or more specificities is one of the most innovative fields in therapeutic antibody development. It has tremendous potential for use in creating new treatments for patients who currently have unmet needs. Bispecific-antibody development also is stimulating innovations in bioprocessing techniques from expression through upstream processing and candidate purification. Wherever possible, process-development scientists and engineers are borrowing techniques honed in mature MAb platforms and applying them to bsAb manufacturing. Nevertheless, the unique qualities of bispecifics make developing biomanufacturing strategies for them a still-emerging art form. Much remains to be done toward creating high-producing and high-yielding processes for this class of products, and challenges are likely to increase as bsAbs become more sophisticated and complex in the future.

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21 Chen SW, Zhang W. Current Trends and Challenges in the Downstream Purification of BsAbs. Antibody Therapeut. 4(2) 2021: 73–88;

22 Tustian AD, et al., Development of a Novel Affinity Chromatography Resin for Platform Purification of BsAbs with Modified Protein A Binding Avidity. Biotechnol. Progr. 24(30) 2018: 650–658;

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Dr. Janice Reichert is editor in chief of mAbs (an Informa Taylor & Francis journal) and executive director of The Antibody Society, Inc., 247 Prospect Street, Framingham MA 01701;; Corresponding author Dr. Nick Hutchinson is deputy chair of The Antibody Society’s communications and membership committee and business steering group lead for mammalian cell culture at Fujifilm Diosynth Biotechnologies, Belasis Avenue, Stockton-on-Tees, Billingham, UK, TS23 1LH;;

This article is abridged from BPI’s September 2021 eBook, “Bispecific Antibodies: Their Development and Manufacturing As Therapeutics” by Janice Reichert and Nick Hutchinson. Download the full version, including pipeline details and illustrative figures, on the BPI website here:

Solving Immunogenicity Problems for Bispecifics by Millie Nelson
This past summer, BioProcess Insider sat down with Zymeworks’s chief scientific officer Tony Polverino to talk about his company’s experiences with developing bispecific antibodies (bsAbs) as cancer treatments. (1).What concerns surround cancer therapies based on bispecific antibodies? I think that the challenges fall into a number of different areas, one area being manufacturing. The [need] to bring two different molecules or two different binding arms together into the same molecule, presents a challenge from an expression and a manufacturing perspective.How have you overcome those issues? Zymeworks has four proprietary platform technologies that make any protein possible, from cytotoxic antitumor biologics (cell-killing compounds) to antibody–drug conjugates (ADCs) and immunooncology therapies.

Our Azymetric platform can be used [for rapid generation of] novel bi- and multispecific therapeutic antibodies [that can] bind multiple cellular targets at one time. That simultaneously enables multiple, important therapeutic outcomes, including stimulation of an immune response, down-regulation of antigen signaling, and growth inhibition or death of diseased cells.

Our ZymeLink technology is a next-generation ADC platform designed to deliver multifunctional, toxic drugs to tumor cells optimally. It overcomes many of the stability and toxicity limitations associated with existing ADCs to realize the full potential of this therapeutic class for patients.

Then there is our EFECT platform (Effector Function Enhancement and Control Technology). It selectively modulates antibody interactions with the immune system to enhance or mute the immune response.

Our newest platform, which we launched earlier this year at the American Association for Cancer Research (AACR) annual meeting, is ProTECT (PROgrammed Tumor Engagement and Checkpoint/Co-stimulation Targeting). It is the first conditionally active antibody technology that simultaneously addresses both ends of the therapeutic window, potentially reducing toxicity and increasing efficacy.

What about flexibility? We can take a variety of different binding arms and put them together very quickly. We can use variants of those binding arms interchangeably and easily.

Why is interchangeability important? Human biology is complex. In binding the same protein, it’s important that both binding arms [on a bispecific antibody] can interact for optimal binding. Sometimes if the two binding arms are too close together, they’re not able to bind efficiently. If they’re too far apart, they’re not able to bind together efficiently either. However, because we have so much flexibility, we can identify the best binding partners and put them together into the same molecule.

Why is it beneficial to have two different binding arms in one molecule? Traditionally, antibodies have had two binding arms that normally target the same protein. It was recognized a long time ago that although that is incredibly valuable — and a lot of drugs have been developed using that basic technology — to improve the activity in different settings, it’s desirable to have two different binding arms together in the same molecule. That’s what our platform technology does. Two different binding arms allow us either to target the same antigen more efficiently or to target two [different] proteins, whether on cancer cells or to bridge cancer cells and immune cells.

What other issues arise with bsAb development? We have recognized that sometimes it’s difficult to get absolute specificity against a tumor cell. A number of therapies in development sometimes suffer from toxicities either because the antigen and the binding domain are not 100% unique to tumor cells or hyperactivation of the immune system leads to some systemic toxicities — similar in some respects to what patients with COVID-19 have experienced.

What is your solution to that problem? We thought about what the problem was, what we needed to solve, and we wanted to solve the problem in two ways. The first question was, “How do we keep the immune system at bay and really focus on the tumor cells as much as possible?” The second question was, “Are there suppressive pathways or other activities that we would like to introduce into our molecule to enhance the efficacy?”

ProTECT is the platform with which we think we’ve demonstrated an ability to [engage] both ends of the therapeutic window.

What enables that platform to address those problems? We’ve enhanced activity and safety. We maximize the therapeutic index with [molecular] binding domains — in particular, the T-cell binding domain — masked by proteins that interact with the immune system, such as checkpoint inhibitors. Those have been successful against a number of different tumors. Unmasking enables binding to immune cells, but the mask itself remains bound and is functional to modulate the immune response additionally. Not only do we think that’s a unique concept, but the platform will have broad applicability. We’ve designed it to maintain flexibility.

Has COVID-19 influenced your way of working? We have established processes and procedures to optimize the development of antibody-based therapeutics. I think COVID made us realize that we can move quickly, if required. Cooperation between governments and companies exemplified how improvements to the system could be implemented. However, the strain on the system overall has introduced challenges for everybody, especially related to the supply chain. Overall, I think COVID affected us just as it did anybody else. Our ability to develop and deliver antibody-based therapeutics is only being strengthened now, and that’s because of the ability that antibodies have to fight disease and delivery of other types of therapies. We need to ensure that we have a stable supply chain and manufacturing base. In addition we have established long range plans with our key contract research organizations (CROs) to prevent any impact on our drug development process.

1 Nelson M. Zymeworks on How to Keep Immune System at Bay When Developing Bispecifics. BioProcess Insider 23 August 2021;

Millie Nelson is a reporter for BioProcess Insider,